Method for inhibiting osteoclast development

ABSTRACT

This invention provides a composition comprising Formula I, or salt thereof, 
                         
wherein X is either chlorine or bromine, Y is either hydrogen or an alkyl group having a carbon chain length from 1 to 5 carbon atoms, and R is an alkyl group having a carbon chain length from 1 to 5 carbon atoms, except that X is not chlorine when Y is hydrogen and R is an ethyl group. A method of preventing bone erosion in a patient and a method of reducing inflammation in a patient using the compositions of Formula I are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This utility patent application claims the benefit of priority to U.S.Provisional Patent Application Ser. No. 61/635,525 filed Apr. 19, 2012.The entire contents of U.S. Provisional Patent Application Ser. No.61/635,525 are incorporated by reference in its entirety into thisutility patent application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. R01 AG012951, R01 AR 053566, R01 ES 011311, R01 AR 065407, and R01 AR 053976awarded by the National Institute of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides a method for inhibiting osteoclastdevelopment and a method for preventing bone erosion by delivering aneffective amount of a haloanilide to an osteoclast for inhibiting theosteoclast development and preventing bone erosion. Preferably, thehaloanilide is N-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide, orN-(3,4-dichlorophenyl)-N-methylisobutyramide.

2. Background Art

The composition N-(3,4-dichlorophenyl)propanamide (“DCPA”) iscommercially available under the tradename “Propanil” for use as aherbicide. Its primary use in to control grassy weeds in rice fieldsbecause rice, as well as wheat, has naturally higher levels ofacylamidase. Acylamidase enzymatically breaks DCPA into dichloroaniline(DCA) and water, thus inactivating the herbicide qualities of the DCPA.DCA is not toxic to plants, and the rice is unharmed but grassy weeds,which have low natural levels of acylamidase, are killed by theherbicide DCPA. Generally, the herbicide DCPA is applied numerous timesduring a growing season, often by spray plane because the rice is grownunder very wet conditions.

The toxicity of DCPA has been investigated. It is actually of lowsystemic toxicity. Mice can tolerate doses in excess of 100 mg/kg andonly doses >150 mg/kg produce frank immunotoxicity. The metabolism ofDCPA by acylamidase produces DCA as mentioned above. DCA is thenmetabolized to N—OH-DCA and 6-OH-DCA. Both N—OH-DCA and 6-OH-DCAcontribute to the systemic toxicity associated with in vivoadministration of DCPA.

It is known that DCPA is anti-inflammatory. DCPA is capable of reducingthe secretion of pro-inflammatory cytokines from macrophages both invivo and ex vivo. The mechanism of the anti-inflammatory effect of DCPAwas first determined in 1997. This mechanism is that DCPA is capable ofinhibiting intracellular calcium release in macrophages.

SUMMARY OF THE INVENTION

This invention provides a composition comprising a haloanilide, or saltthereof, that is a selective inhibitor of a Ca²⁺ release activated Ca²⁺(CRAC) channel, wherein the haloanilide composition is notN-(3,4-dichlorophenyl)propanamide.

In a preferable embodiment of this invention, compositions of thisinvention are provided comprising Formula I, or a salt thereof,

wherein X is either chlorine or bromine, Y is either hydrogen or analkyl group having a carbon chain length from 1 to 5 carbon atoms, and Ris an alkyl group having a carbon chain length from 1 to 5 carbon atoms,except that wherein X is not chlorine when Y is hydrogen and R is anethyl group. More preferably, the compositions of this invention includewherein the alkyl group has from three to five carbon atoms and thecarbon atoms are in either a straight chain or a branch chainarrangement. The compositions of Formula I preferably include wherein Ris an ethyl group or wherein R is an isopropyl group. Most preferably,the compositions of this invention are selected from the groupconsisting of N-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide [also referred to as “DNI”], andN-(3,4-dichlorophenyl)-N-methylisobutyramide.

Another embodiment of this invention provides a method of inhibitingosteoclast development comprising administering an effective amount of ahaloanilide composition of this invention or a salt thereof, to anosteoclast cell for inhibiting osteoclast development, wherein thehaloanilide composition is not N-(3,4-dichlorophenyl)propanamide(“DCPA”). A preferred embodiment of this invention provides wherein thehaloanilide composition of this invention is a composition of Formula I,or salt thereof, comprising

wherein X is either chlorine or bromine, Y is either hydrogen or analkyl group having a carbon chain length from 1 to 5 carbon atoms, and Ris an alkyl group having a carbon chain length from 1 to 5 carbon atoms,except wherein X is not chlorine when Y is hydrogen and R is an ethylgroup. More preferably, this method as described herein, includeswherein the alkyl group has from three to five carbon atoms and thecarbon atoms are in either a straight chain or a branch chainarrangement. This method of this invention, as described herein,preferably includes wherein R is an ethyl group, or wherein R is anisopropyl group. Most preferably, this method includes wherein Formula Iis a composition selected from the group consisting ofN-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide [also referred to as “DNI”], andN-(3,4-dichlorophenyl)-N-methylisobutyramide.

Another embodiment of the present invention provides a method forpreventing bone erosion, especially in a patient diagnosed witharthritis, comprising administering to a patient an effective amount ofa haloanilide composition of this invention, or a salt thereof, forpreventing bone erosion, especially due to arthritis, in a patient,wherein the haloanilide composition is notN-(3,4-dichlorophenyl)propanamide. Preferably, this method includeswherein the haloanilide is a composition of Formula I, or salt thereof,comprising

wherein X is either chlorine or bromine, Y is either hydrogen or analkyl group having a carbon chain length from 1 to 5 carbon atoms, and Ris an alkyl group having a carbon chain length from 1 to 5 carbon atoms,wherein X is not chlorine then Y is hydrogen and R is an ethyl group,except wherein X is not chlorine when Y is hydrogen and R is an ethylgroup. More preferably, this method, as described herein, includeswherein the alkyl group has from three to five carbon atoms and thecarbon atoms are in either a straight chain or a branch chainarrangement. This method of this invention preferably includes whereinthe R is an ethyl group or an isopropyl group. Most preferably, thismethod, as described herein, includes wherein Formula I is a compositionselected from the group consisting of N-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide [also referred to as “DNI”], andN-(3,4-dichlorophenyl)-N-methylisobutyramide.

Another embodiment of this invention provides a method of inhibiting aCa²⁺ release activated Ca²⁺ (CRAC) channel either in vivo or in vitrocomprising subjecting a cell derived from a blood monocyte to aneffective amount of a haloanilide composition of this invention, or saltthereof, for inhibiting a Ca²⁺ release activated Ca²⁺ (CRAC) channel ofthe cell, wherein the haloanilide composition is notN-(3,4-dichlorophenyl)propanamide. In a preferred embodiment of thismethod, as described herein, the haloanilide is a composition of FormulaI, or salt thereof, comprising

wherein X is either chlorine or bromine, Y is either hydrogen or analkyl group having a carbon chain length from 1 to 5 carbon atoms, and Ris an alkyl group having a carbon chain length from 1 to 5 carbon atoms,except wherein X is not chlorine when Y is hydrogen and R is an ethylgroup. More preferably, the method includes wherein the alkyl group hasfrom three to five carbon atoms and the carbon atoms are in either astraight chain or a branch chain arrangement. This method, as describedherein, preferably includes wherein R is an ethyl group or an isopropylgroup. Most preferably, this method, as described herein, includeswherein the haloanilide is composition selected from the groupconsisting of N-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide [also referred to as “DNI”], andN-(3,4-dichlorophenyl)-N-methylisobutyramide.

In another embodiment of this invention, a method of reducinginflammation in a patient is provided comprising administering to apatient an effective amount of a haloanilide composition or saltthereof, for reducing inflammation in a patient, wherein the haloanilidecomposition is not N-(3,4-dichlorophenyl)propanamide. Preferably, thismethod of reducing inflammation in a patient includes wherein thehaloanilide is a composition of Formula I, or salt thereof, comprising

wherein X is either chlorine or bromine, Y is either hydrogen or analkyl group having a carbon chain length from 1 to 5 carbon atoms, and Ris an alkyl group having a carbon chain length from 1 to 5 carbon atoms,wherein X is not chlorine then Y is hydrogen and R is an ethyl group,except wherein X is not chlorine when Y is hydrogen and R is an ethylgroup. More preferably, this method of reducing inflammation in apatient includes wherein the alkyl group has from three to five carbonatoms and the carbon atoms are in either a straight chain or a branchchain arrangement. More preferably, this method of reducing inflammationin a patient includes wherein the R is either an ethyl group or anisopropyl group. Most preferably, this method of reducing inflammationcomprises administering to a patient an effective amount of thecomposition of Formula I that is selected from the group consisting ofN-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide, andN-(3,4-dichlorophenyl)-N-methylisobutyramide, for reducing inflammationin the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E show modulation of store-operated calcium channels duringosteoclast differentiation. FIGS. 1A-E show store operated calcium entry(SOCe) measured in monocytes maintained in m-CSF. The insets of FIGS.1A-E depict each individual cell within the boxed regions to reveal Ca²⁺fluxes during the period prior to the addition of Tg. FIG. 1F shows thepercentage of cells exhibiting Ca²⁺ fluxes as depicted in FIGS. 1A-E.FIG. 1G shows the total amount of store-operated Ca²⁺ entry at each timepoint. FIG. 1H is a Western Blots for STIM I, STIM2, and Orai I andActin in isolated monocytes maintained in m-CSF (day zero) andsupplemented with RANKL for 1, 3, 7, or 11 days.

FIGS. 2A-2E show siRNA knockdown of Orai inhibits human osteoclastdevelopments in vitro. FIG. 2A shows transfection of a mixture of foursiRNAs to reduce Orai I expression. FIG. 2B shows a Western Blot siRNAsOrai I protein after three days of transfection relative to controlstransfected with scrambled siRNA. FIG. 2C shows Orai I mRNA quantifiedin transfected and control cells relative to GAPDH by use ofquantitative real-time PCR as a function of time. After three days, mRNAis reduced but the siRNA is progressively lost. FIG. 2D shows treatmentof cell cultures for 7 days with RANKL relative to the same mediumwithout RANKL did not affect Orai I mRNA level relative to GAPDH, whichsuggest that expression is not down-regulated by osteoclastdifferentiation. FIG. 2E shows cells with Orai I knocked down producefew nucleated cells. Cells were maintained in osteoclast differentiationmedium with RANKL and m-CSF, for seven days after transfection.Multinucleated cells were reduced about 70% by knockdown and very fewcells with more than three nuclei were present (black bars) relative tocontrol (gray bars).

FIG. 3 shows the known chemical structure ofN-(3,4-dichlorophenyl)propionamide, also known as “DCPA” or“N-(3,4-dichlorophenyl)propanamide” or “PROPANIL”.

FIG. 4 shows the chemical structures of the more preferred haloanilidecompositions of the present invention, namely,N-(3,4-dibromophenyl)propionamide (also known as “DBPA” or“N-(3,4-dibromophenyl)propanamide”), andN-(3,4-dichlorophenyl)-N-methylpropionamide (also known as“N,N-Me,Propyl-CA” or “N-Methyl-DCPA” or “NMP” or“N-(3,4-dichlorophenyl)-N-methylpropanamide”).

FIG. 5 shows the chemical structures of the more preferred haloanilidecompositions of the present invention, namely,N-(3,4-dichlorophenyl)isobutylamide (also known as “N-Isobutyl-CA” or“DNI”), and N-(3,4-dichlorophenyl-N-methylisobutyramide (also known as“N,N-Me,Isobutyl-CA”).

FIG. 6 shows a graph having the toxicity results of DCPA; DBPA;N,N-Me,Propyl-CA; N-Isobutyl-CA; N,N-Me, Isobutyl-CA; and ethanolcontrol on Jurkat cells exposed 48 hours to each of these compositions,respectively, at the concentrations indicated on the x-axis of thegraph, and analyzed by flow cytometry using the far-red fluorescent DNAbinding probe 7-aminoactinomycin D (i.e. 7-AAD) to indicate dead or lateapoptotic cells indicated on the y-axis of the graph.

FIG. 7 shows synthesis scheme for the compositions of the presentinvention.

FIG. 8 shows inhibition of CRAC channels with a composition of thepresent invention, namely, DBPA [ie. N-(3,4-dibromophenyl)propanamide].

FIG. 9 shows inhibition of CRAC channels with compositions of thepresent invention, namely, DNI, NM Propanil, and DNMNI.

FIG. 10A shows the dose response of a composition of the presentinvention, namely, N-(3,4-dichlorophenyl)-N-methylpropanamide (alsoknown as “NMP” or “N-methyl DCPA”) at doses of 3 μM, 10 μM, 15 μM, 60μM, 100 μM, 150 μM, 300 μM, and 1 mM, respectively. FIG. 10B showsStore-Operated Calcium Entry (SOCE) inhibition of NMP having a IC₅₀ of104.2 μM.

DETAILED DESCRIPTION OF THE INVENTION

The present invention investigated the effect ofN-(3,4-dichlorophenyl)propanamide (“DCPA”) on calcium influx in a T cellline as well as HEK cells (fibroblast cell line often used for cellularphysiology). The calcium (Ca²⁺) release activated calcium (Ca²⁺)(“CRAC”) channel is fully characterized in T cells. The presentinvention shows that DCPA inhibits CRAC channel activity which preventscalcium influx necessary for T cell activation (cytokine production).DCPA inhibits calcium influx in macrophages. Macrophages are tissuebound cells that are derived from blood monocytes under the influence ofspecific growth factors and cytokines. Other cell types that are alsoderived from blood monocytes include osteoclasts. Osteoclasts destroybone and are one of two main cells that maintain the normal homeostasisof bones. Bone is not a static tissue and there is a constant breakdownof bone by osteoclasts balanced with new bone formation by osteoblasts.Patients with arthritis (osteoarthritis and rheumatoid arthritis) havean imbalance of this process and there is an excess of osteoclastactivity with leads to joint deformity. Heretofore, there was notreatment available to prevent this excess bone erosion. The presentinvention shows that haloanilide compositions, and their salts, andpreferably haloanilides selected from the group consisting ofN-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide, andN-(3,4-dichlorophenyl)-N-methylisobutyramide, inhibits osteoclastproduction from human blood monocytes in a concentration-dependentmanner. The present invention shows that osteoclasts also utilize CRACchannels for activation and that the haloanilide compositions of thisinvention inhibit CRAC activity in monocytes and that the key activitythat required CRAC activity was forming the multinuclear syncytiacharacteristic of osteoclasts. The present invention shows that thehaloanilide compositions of this invention inhibit CRAC activity bypreventing the formation of punctae by a substructure of the CRACchannel called STIM1. The present invention shows that haloanilidecompositions such as for example but not limited toN-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide, andN-(3,4-dichlorophenyl)-N-methylisobutyramide, inhibit the formation ofcollagen-induced arthritis (CIA) in mice dramatically. CIA is the mostcommonly used animal model for arthritis. The haloanilide compositions,and salts thereof, of this invention, inhibit CRAC channel activity andmay be administered to patients to prevent bone erosion associated witharthritis. Other known CRAC channel inhibitors are far too toxic to beconsidered drug candidates.

The metabolism of DCPA by acylamidase produces DCA as mentioned above.DCA is then metabolized to N—OH-DCA and 6-OH-DCA. Both N—OH-DCA and6-OH-DCA contribute to the systemic toxicity associated with in vivoadministration of DCPA. However, the present inventor has found that theeffect of DCA, N—OH-DCA and 6-OH-DCA on intracellular calcium influx andtheir toxicity was not associated with CRAC channel inhibition. Thus,the CRAC channel inhibitory activity of DCPA is not attributable to anyof these toxic metabolites. Given that the CRAC channel inhibitionassociated with DCPA was not due to the more toxic metabolites, thepresent invention discloses haloanilide compositions of Formula I, andsalts thereof that are not metabolized to DCA but that have similar CRACchannel inhibitory qualities of DCPA:

wherein X is either chlorine or bromine, Y is either hydrogen or analkyl group having a carbon chain length from 1 to 5 carbon atoms, and Ris an alkyl group having a carbon chain length from 1 to 5 carbon atoms,except wherein X is not chlorine when Y is hydrogen and R is an ethylgroup. Synthesis routes for N-(3,4-dibromophenyl)propanamide [alsoreferred to as “DBPA” or “N-(3,4-bibromophenyl)propionamide”],N-(3,4-dichlorophenyl)-N-methylpropanamide [also referred to as“N-Methyl Propanil”; or “N-Methyl-DCPA”; or “NMP”; or “NM Propanil”; or“N-(3,4-dichlorophenyl)-N-methylpropionamide”; or “N,N-Methyl,Propyl-CA” (CA=carboxamide)], 3,4-dichloro-N-methyl-N-isobutyramide[also referred to as “N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide”;or “DNMI”], N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide [also referred to as “DNI” or“N-Isobutyl-CA” (CA=carboxamide)], andN-(3,4-dichlorophenyl)-N-methylisobutyramide [also referred to as“N,N-Me, Isobutyl-CA” (CA=carboxamide)]. Each of the haloanilidecompositions of this invention have undergone in vitro toxicity testingon Jurkat cells—this is a T cell line that is sensitivity to toxiceffects and thus, was used to determine the effect of the compounds onviability. Each of the compositions of Formula I are capable ofinhibiting CRAC channel activity as measured by intracellular calciuminflux. It was found that N-methyl-DCPA is effective at lowerconcentrations. FIGS. 4 and 5 show chemical structures of preferredhaloanilide compositions of this invention.

Another embodiment of this invention provides a method of inhibitingosteoclast development comprising administering an effective amount of ahaloanilide composition of this invention or a salt thereof, to anosteoclast cell for inhibiting osteoclast development, wherein thehaloanilide composition is not N-(3,4-dichlorophenyl)propanamide(“DCPA”). A preferred embodiment of this invention provides wherein thehaloanilide composition of this invention is a composition of Formula I,or salt thereof, comprising

wherein X is either chlorine or bromine, Y is either hydrogen or analkyl group having a carbon chain length from 1 to 5 carbon atoms, and Ris an alkyl group having a carbon chain length from 1 to 5 carbon atoms,except wherein X is not chlorine when Y is hydrogen and R is an ethylgroup. More preferably, this method as described herein includes whereinthe alkyl group has from three to five carbon atoms and the carbon atomsare in either a straight chain or a branch chain arrangement. Thismethod of this invention, as described herein, preferably includeswherein R is an ethyl group, or wherein R is an isopropyl group. Mostpreferably, this method includes wherein Formula I is a compositionselected from the group consisting of N-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide [also referred to as “DNI”], andN-(3,4-dichlorophenyl)-N-methylisobutyramide.

Another embodiment of the present invention provides a method forpreventing bone erosion in a patient diagnosed with arthritis comprisingadministering to a patient an effective amount of a haloanilidecomposition of this invention, or a salt thereof, for preventing boneerosion in a patient, wherein the haloanilide composition is notN-(3,4-dichlorophenyl)propanamide. Preferably, this method of preventingbone erosion, of this invention, includes wherein the haloanilide is acomposition of Formula I, or salt thereof, comprising

wherein X is either chlorine or bromine, Y is either hydrogen or analkyl group having a carbon chain length from 1 to 5 carbon atoms, and Ris an alkyl group having a carbon chain length from 1 to 5 carbon atoms,wherein X is not chlorine then Y is hydrogen and R is an ethyl group,except wherein X is not chlorine when Y is hydrogen and R is an ethylgroup. More preferably, this method, as described herein, includeswherein the alkyl group has from three to five carbon atoms and thecarbon atoms are in either a straight chain or a branch chainarrangement. Preferably, this method of this invention includes whereinthe R is an ethyl group or an isopropyl group. Most preferably, thismethod, as described herein, includes wherein Formula I is a compositionselected from the group consisting of N-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide [also referred to as “DNI”], andN-(3,4-dichlorophenyl)-N-methylisobutyramide.

Another embodiment of this invention provides a method of inhibiting aCa²⁺ release activated Ca²⁺ (CRAC) channel either in vivo or in vitrocomprising subjecting a cell derived from a blood monocyte to aneffective amount of a haloanilide composition of this invention, or saltthereof, for inhibiting a Ca²⁺ release activated Ca²⁺ (CRAC) channel ofthe cell, wherein the haloanilide composition is notN-(3,4-dichlorophenyl)propanamide. In a preferred embodiment of thismethod, as described herein, the haloanilide is a composition of FormulaI, or salt thereof, comprising

wherein X is either chlorine or bromine, Y is either hydrogen or analkyl group having a carbon chain length from 1 to 5 carbon atoms, and Ris an alkyl group having a carbon chain length from 1 to 5 carbon atoms,except wherein X is not chlorine when Y is hydrogen and R is an ethylgroup. More preferably, the method includes wherein the alkyl group hasfrom three to five carbon atoms and the carbon atoms are in either astraight chain or a branch chain arrangement. This method, as describedherein, preferably includes wherein R is an ethyl group or an isopropylgroup. Most preferably, this method, as described herein includeswherein the haloanilide is composition selected from the groupconsisting of N-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide [also referred to as “DNI”],N-(3,4-dichlorophenyl)-N-methylisobutyramide.

Yet another embodiment of this invention provides a compositioncomprising a haloanilide, or salt thereof, that is a selective inhibitorof a Ca²⁺ release activated Ca²⁺ (CRAC) channel, wherein the haloanilidecomposition is not N-(3,4-dichlorophenyl)propanamide. Preferably, thecompositions of this invention comprise Formula I, or salt thereof,

wherein X is either chlorine or bromine, Y is either hydrogen or analkyl group having a carbon chain length from 1 to 5 carbon atoms, and Ris an alkyl group having a carbon chain length from 1 to 5 carbon atoms,except that wherein X is not chlorine when Y is hydrogen and R is anethyl group. More preferably, the compositions of this invention includewherein the alkyl group has from three to five carbon atoms and thecarbon atoms are in either a straight chain or a branch chainarrangement. The compositions of Formula I preferably include wherein Ris an ethyl group or wherein R is an isopropyl group. Most preferably,the compositions of this invention are selected from the groupconsisting of N-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide [also referred to as “DNI”],N-(3,4-dichlorophenyl)-N-methylisobutyramide.

Osteoclasts are specialized macrophage derivatives that secrete acid andproteinases to mobilize bone for mineral homeostasis, growth, andreplacement or repair. Osteoclast differentiation generally requires themonocyte growth factor m-CSF and the TNF-family cytokine RANKL, althoughdifferentiation is regulated by many other cytokines and byintracellular signals, including Ca²⁺. Studies of osteoclastdifferentiation in vitro were performed using human monocytic precursorsstimulated with m-CSF and RANKL, revealing significant loss in both theexpression and function of the required components of store-operatedCa²⁺ entry over the course of osteoclast differentiation. However,inhibition of CRAC using either the pharmacological agent3,4-dichloropropioanilide (DCPA) or by knockdown of Orai I severelyinhibited formation of multinucleated osteoclasts. In contrast, noeffect of CRAC channel inhibition was observed on expression of theosteoclast protein tartrate resistant acid phosphatase (TRAP). Findingssuggest that despite the fact that they are down-regulated duringosteoclast differentiation, CRAC channels are required for cell fusion,a late event in osteoclast differentiation. Since osteoclasts cannotfunction properly without multinucleation, selective CRAC inhibitors mayhave utility in management of hyperresorptive states (see, Zhou et al.,J. Cell. Physiol., Vol. 226, pages 1082-1089, 2011). It is known thatosteoclast differentiation from monocytes and regulation of attachmentto bone are dependent on inositol 1,4,5-trisphosphate (InsP₃)-mediatedCa²⁺-release from the endoplasmic reticulum (ER). Less clear is theextent to which extracellular Ca²⁺ influx is involved osteoclastdifferentiation. In hematopoietic cells, it is known thatCa²⁺-release-activated Ca²⁺ (CRAC) channel activity represents the majormeans of Ca⁺ entry. Further, the type IA transmembrane protein STIMI andthe plasma membrane Ca²⁺ channel Orai I have been defined as themolecular mediators of CRAC channel activity.

In CRAC channel activation, the ER luminal portion of the activatingprotein STIM I, which contains a low-affinity Ca²⁺-binding EF handmediates activation of Orai I at the cell membrane. In this process, ERCa²⁺ release, via InsP₃ receptors typically, causes a STIM Iconformational change that causes STIM I aggregation at sites adjacentto the plasma membrane commonly referred to as puncta. At puncta thecytoplasmic membrane portion of STIM I physically interacts with theplasma membrane-localized Ca²⁺ channel Orai I resulting in itsactivation. The extent to which this pathway is active during osteoclastdifferentiation is not established.

The present inventor has studied Orai I and STIM I expression andfunction during osteoclast differentiation in vitro, and has foundsignificant decreases early in the process of osteoclastdifferentiation. Further, reduction of Orai I expression with siRNAinhibited osteoclast differentiation, particularly multinucleation.Intriguingly, the addition of the pharmacological agent3,4-dichloropropioanilide (DCPA) similarly inhibited terminal osteoclastdifferentiation. DCPA-mediated CRAC channel inhibition occurs viainhibition of STIMI-Orai I interaction. DCPA is a haloanilide compoundwith relatively low systemic toxicity that can inhibit Ca⁺ influx inmacrophages and T cells as well as having potent anti-inflammatoryactivity.

In another embodiment of this invention, a method of reducinginflammation in a patient is provided comprising administering to apatient an effective amount of a haloanilide composition or saltthereof, for reducing inflammation in a patient, wherein the haloanilidecomposition is not N-(3,4-dichlorophenyl)propanamide. Preferably, thismethod of reducing inflammation in a patient includes wherein thehaloanilide is a composition of Formula I, or salt thereof, comprising

wherein X is either chlorine or bromine, Y is either hydrogen or analkyl group having a carbon chain length from 1 to 5 carbon atoms, and Ris an alkyl group having a carbon chain length from 1 to 5 carbon atoms,wherein X is not chlorine then Y is hydrogen and R is an ethyl group,except wherein X is not chlorine when Y is hydrogen and R is an ethylgroup. More preferably, this method of reducing inflammation in apatient includes wherein the alkyl group has from three to five carbonatoms and the carbon atoms are in either a straight chain or a branchchain arrangement. More preferably, this method of reducing inflammationin a patient includes wherein the R is either an ethyl group or anisopropyl group. Most preferably, this method of reducing inflammationcomprises administering to a patient an effective amount of thecomposition of Formula I that is selected from the group consisting ofN-(3,4-dibromophenyl)propanamide,N-(3,4-dichlorophenyl)-N-methylpropanamide,N-(3,4-dichlorophenyl)-N,2-dimethylpropanamide),N-(3,4-dichlorophenyl)-2-dimethylpropanamide,N-(3,4-dichlorophenyl)isobutyramide, andN-(3,4-dichlorophenyl)-N-methylisobutyramide, for reducing inflammationin the patient.Materials and MethodsCell Culture, Cell Fines, and Cell Differentiation In Vitro

Human monocytes were isolated from normal buffy coat cells (60-80 ml)obtained with approval of institutional review boards by separation fromdonor blood, the white-cell depleted blood retained for clinical use.Human monocyte culture and differentiation were as reported (see,Yaroslayskly B B, Zhang Y, Kalla S E, Garcia P V, Sharrow A C, Li Y,Zaidi M, Wu C, Blair H C, “NO-dependent osteoclast motility: Reliance oncGMP-dependent protein kinase I and VASP”, J Cell Sci I, Vol. 18, pages5479-5487, 2005). Briefly, human monocytes cells were isolated frombuffy coat on ficoll lymphocyte separation media and cultured at ˜6×10⁵cells per cm² in monocyte maintenance medium, Dulbecco's modifiedessential medium (DMEM) with 20 ng/ml of human m-CSF and 10% FBS, for 24hours (h). After 24 h in culture, the medium was changed to osteoclastdifferentiation medium containing in addition of human 50 ng/ml ofRANKL, with the additional inhibitors or activators as specified inresults. Human osteoclast cultures were generally maintained for aminimum of 7 days before analysis, or longer times as specified inresults. Characterization of osteoclasts included in situ demonstrationof tartrate resistant acid phosphatase (TRAP) using naphthol phosphatesubstrate coupled with fast garnet at pH 5 in 200 mM tartrate (leucocyteacid phosphatase, Sigma-Aldrich, St. Louis, Mo.) and by evaluation ofmultinucleation using either phase contrast microscopy or nuclearstains.

Western Blots

Cells were lysed in 1% NP-40 (nonyl phenoxylpolyethoxylethanol), 150 mMNaCl, 50 mM Tris, pH 8.0, with proteinase inhibitors, cleared bycentrifugation, and normalized for protein, determined by Bradforddye-binding. Proteins were resolved by electrophoresis on 8%polyacrylamide in sulfonyl dodecyl sulfate, and transferred topolyvinylidene difluoride derivitized nylon. Membranes were blocked inTris-buffered saline with 0.05% polyoxyethylene sorbitan (Tween 20) with5% bovine serum albumin, 1 h, 20° C. (centigrade), and incubated withthe primary antibodies at 4° C. overnight. Membranes were washed and theappropriate peroxidase-conjugated secondary antibody added. After a30-min (minute) incubation, membranes were washed and bands werevisualized by enhanced chemiluminescence (ECL-Western Blot Reagent Kit,GE Healthcare, Waukesha, Wis.).

Electro Physiology

Analysis was performed in HEK293 cells stably expressing Orai I andtransfected with STIM I YFP used conventional whole cell voltagerecordings as described (see, Wang Y, Deng X, Zhou Y, Hendron E,Mancarelia S, Ritchie M F. Tang X D, Baba Y, Kurosaki T, Mori Y,Soboloff J, Gill D L., “STIM protein coupling in the activation of Oraichannels”, Proc Natl Acad Sci USA Vol. 106, pages 7391-7396, 2009).Immediately after establishment of the whole-cell electrode seal,voltage ramps spanning from—100 to +100 mV in 50 msec were deliveredfrom a holding potential of 0 mV at a rate of 0.5 Hz. A 10 mV junctionpotential compensation was applied. The intracellular solution contained145 mM CsGlu, 10 mM HEPES, 10 mM EGTA, 8 mM NaCl, 6 mM MgCl₂, and 2 mMMg-ATP (total 8 mM Mg²⁺), pH 7.2; TRPM7 activity was suppressed by 8 mMMg²⁺ and ATP (see, Zhou Y, Mancarella S, Wang Y, Yue C, Ritchie M, GillD L, Soboloff J, “The short N-terminal domains of STIM I and STIM2control the activation kinetics of Orai I channels”, J Biol Chem, Vol.284, pages 19164-19168, 2009). The extracellular solution contained 145mM NaCl, 10 mM CaCl₂, 10 mM CsCl, 2 mM MgCl₂, 2.8 mM KCl, 10 mM HEPES,and 10 mM glucose, pH 7.4.

DNA and RNA Reagents

PCR Primers:

Homo sapiens Ca²⁺ release-activated Ca²⁺ modulator I (ORAI I) NM_032790,commercially available from OriGene, Rockville, Md.

Homo sapiens glyceraldehyde-3-phosphate dehydrogenase (GAPDH) NM_002046,commercially available form OriGene, Rockville, Md.

Orai I silencing used a pool of four siRNAs targeting Homo sapiens ORAII (GenBank NM_032790), purchased as a pretested reagent from DharmaconRNAi Technologies (smartpool 84876, Thermo-Fisher, Waltham, Mass.).Cells were transfected using siPORTAmine (Ambion, Austin, Tex.), a blendof polyamines, as described (see, Yaroslayskly et al., 2005, above).Controls were transfected with nonsense siRNA.

To visualize transfection, Cy5 was covalently attached to the duplexsiRNA (Ambion Silencer siRNA labeling kit). mRNA was quantified byreal-time PCR as described in Robinson L j, Yaroslayskly B B, Griswold RD, Zadorozny E V, Guo L, Tonrkova I L, Blair H C, “Estrogen inhibitsRANKL-stimulated osteoclastic differentiation of human monocytes throughestrogen and RANKL-regulated interaction of estrogen receptor-alpha withBCARI and Traf6”, Exp Cell Res, Vol. 315, pages 1287-1301, 2009.

STIM I Puncta Formation

HEK293 cells, maintained in DMEM with 10% FBS, were transfected withYFP-STIM I using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) for 5h (37° C.; 5% CO₂) followed by a 24-h recovery. Cells were placed in 140mM NaCl, 5 mM KCl, 1 mM MgCl₂, 10 mM glucose, 15 mM HEPES, 0.1% BSA, 2mM CaCl₂ and analyzed by confocal microscopy for STIM I puncta formationusing a Nikon phase-fluorescence microscope with CCD detectors(Diagnostic instruments, Sterling Heights, Mich.). Phase or transmittedlight microscopy used 10-40× objectives and red, green, and blue filtersto assemble color images. Fluorescence images used 1.4 NA 40× oilobjectives. Red fluorescence used excitation at 530-560 nm, a 575 nmdichroic minor, and 580-650 nm emission filter. Transfection data wereanalyzed using Nikon NIS Elements Imaging Software. The effect of DCPAon STIM I/Orai I association was determined in wild-type HEK293 cellstransfected with STIM I-YFP as described in Wang Y, Deng X, Zhou Y,Hendron E, Mancarelia S, Ritchie M F, Tang X D, Baba Y, Kurosaki T, MoriY, Soboloff J, Gill DL, “STIM protein coupling in the activation of Oraichannels”, Proc Natl Acad Sci USA, Vol. 106, pages 7391-7396, 2009.Experiments were performed on a Leica DMI 6000B fluorescence microscopecontrolled by Slidebook Software (Intelligent Imaging Innovations,Denver, Colo.).

Cytosolic Ca²⁺ Measurement

Ratiometric imaging of intracellular Ca²⁺ using fura-2 was as described(see, Zhou et al., 2009 above). Briefly, cells on coverslips, in cationsafe solution (107 mM NaCl, 7.2 mM KCl, 1.2 mM MgCl₂, 11.5 mM glucose,20 mM HEPES-NaOH, pH 7.2) were loaded with fura-2 acetoxymethyl ester (2μM) for 30 min at 24° C. Cells were washed and fluorescent probe wasallowed to de-esterify for 30 min. From signal remaining after saponinpermeabilization, ˜85% of the dye was confined to the cytoplasm (see, MaH-T, Patterson R L. van Rossum D B. Birnbaumer L, Mikoshiba K, Gill D L,“Requirement of the inositol trisphosphate receptor for activation ofstore-operated Ca²⁺ channels”, Science, Vol. 287, pages 1647-1651,2000). Ca²⁺ measurements were made using a Leica DMI 6000B fluorescencemicroscope controlled by Slidebook Software. Fluorescence emission at505 nm was monitored while alternating between 340 and 380 nm excitationwavelengths at a frequency of 0.67 Hz; intracellular Ca²⁺ measurementsare shown as 340/380 nm emission ratios obtained from groups (35-45) ofsingle cells. Measurements shown are representative a minimum of threeindependent experiments.

Materials

The inhibitor DCPA was from ChemServices (West Chester, Pa.). Workingsolutions were made in ethanol at 1,000× final concentration, with equalethanol added to controls. The inactive congener3,4-difluoropropioanilide was synthesized by fluorination ofpropionanilide methyl ester; the product was de-esterified and purifiedby column chromatography, with identification of the purified product byspectroscopy.

Results

Ca²⁺ Homeostasis During Osteoclast Differentiation

Store-dependent and store-independent changes in cytosolic Ca²⁺concentration were examined in human monocytes isolated from buffy coatas they differentiated into osteoclasts in vitro (FIG. 1A-1G). Culturesmaintained in m-CSF were treated with RANKL to induce and supportosteoclast differentiation for 1, 3, 7, or 11 days. At each of thesetime points, cultures were loaded with Fura 2 to measure basal cytosolicCa²⁺ concentration (FIG. 1A-1G) and samples were collected for Westernanalysis (FIG. 1H). Intriguingly, irrespective of the presence ofextracellular Ca²⁺, spontaneous Ca²⁺ fluxes were observed in asignificant percentage of these cells (FIG. 1A-1E). Further, thepercentage of cells exhibiting these spontaneous Ca²⁺ fluxes increasedthe longer cells were maintained in RANKL, reaching as high as 75% atday 11 (FIG. 1F). To assess the capacity of these cells forstore-operated Ca²⁺ entry, they were then treated with theSarco/Endoplasmic Reticulum (SERCA) inhibitor thapsigargin in theabsence of extracellular Ca²⁺ to deplete ER Ca²⁺ content. The subsequentaddition of 1 mM Ca²⁺ revealed significant decreases in the amount ofCa²⁺ entry between 1 and 11 days after the addition of RANKL (FIG.1A-1E, and FIG. 1G). Interestingly, this decrease in the capacity forstore-operated Ca²⁺ entry during osteoclast differentiation coincidedwith decreases in the expression of STIMI, STIM2 and Orai I (FIG. 1H).Hence, not only does RANKL induce Ca²⁺ oscillations, but also thisinvention reveals dramatic changes in both the expression and functionof proteins involved in store-operated Ca²⁺ entry during RANKL-inducedosteoclast differentiation.

Reduced Expression of Orai I Reduces Multinucleation of HumanOsteoclasts In Vitro

To assess the contributions of CRAC channels towards osteoclastdifferentiation, human monocytes were treated with CyS-labeled OraiisiRNA and differentiated in vitro into osteoclasts. Transfectionefficiency with an siRNA cocktail was ˜75% (FIG. 2A), with the ˜80%decrease in Orai I protein by Western analysis (FIG. 2B) suggesting thatvery little Orai I was present in cells that were transfected. Orai ImRNA was also measured in cells at the time of plating and after RANKLaddition for 3, 7, and 11 days (FIG. 2C). Orai I mRNA relative to GAPDHwas reduced 60% at day 3, but message levels increased overtime, inkeeping with loss of siRNA. Irrespective, transfection with Orai I siRNAresulted in a 58% decrease in SOCe relative to control 11 days afterRANKL addition. Interestingly, addition of RANKL had no effect on Orai Iat the RNA level (FIG. 2D), distinct from what was observed for Orai Iprotein expression (FIG. 1H). Nevertheless, knockdown of Orai I markedlyreduced the number of multinucleated cells after 7 days (FIG. 2E,histogram and arrows in middle photomicrograph); multinucleated syncytiaare required for efficient bone degradation and reduced multinucleatedcells are a characteristic of osteopetrosis. However, other propertiesof osteoclasts include induction of TRAP and TRAP activity was similarin control and Orai knockdown cultures (FIG. 2E, arrowheads inphotomicrograph), suggesting that the reduction in multinucleation isdistal to induction of key osteoclast proteins.

Pharmacological Inhibition of Store-Operated Calcium Entry (SOCe)Reduces Multinucleation of Human Osteoclasts

The haloanilide DCPA blocks store-operated Ca²⁺ channel function inJurkat cells with no effect on ER Ca²⁺ content. DCPA also reduced SOCein osteoclasts by 27.5% (n=3; data not shown). Whether effects of DCPAresult from direct modulation of store-operated Ca²⁺ channels was notknown. To address this, the present inventor examined HEK293 cellsstably expressing Orai I and transiently transfected with YFP-STIMI andmeasured the effect of DCPA on CRAC current or I_(crac). CRAC channelactivity was measured in the whole cell clamp position after passivelydepleting Ca²⁺ stores via the presence of 10 mM EGTA in the patchpipette. In both control and DCPA-treated cells, CRAC current began todevelop within 30 sec of break-in. However, CRAC currents began to closeprior to reaching the peak in DCPA-treated cells, returning to baselineless than 4 minutes after break-in. Analysis of the current-voltagerelationship between control and DCPA-treated cells did not showsignificant differences. These observations demonstrate that DCPA is abona-fide CRAC channel inhibitor, although its mechanism of action wasnot yet defined.

The effect of DCPA on human osteoclast differentiation 8 days afteraddition of RANKL was evaluated. Osteoclast differentiation isdemonstrated by multinucleated (>2 nuclei/cell) cells and TRAP activity.DCPA was added at 1.0, 10, and 100 μM to identical cultures at the timeof RANKL addition; 1.0 μM DCPA had minimal effect on the number ofmultinucleated TRAP⁺ cells, but there was a marked progressive reductionin the number of multinucleated cells at 10 and 100 μM DCPA. Despitequalitative differences in cell clumping and other secondary features ofthe cultures, no differences in the TRAP activity were observed even atthe highest concentration of inhibitor, in keeping with results of Oraiknockdown (FIG. 2). The inactive DCPA congener3,4-difluororopropioanilide [also referred to as “DFPA” or“3,4-difluoropropanamide”) had no significant effect on multinucleationat either 10 μM or 100 μM, indicating that DCPA-mediated inhibition ofosteoclast formation results from CRAC inhibition.

DCPA Reduces Puncta Formation by CRAC Channel Components in Wild-TypeHEK293 Cells or HEK293 Cells Overexpressing STIMI and Orai I

Based on the investigation above, it was found that DCPA inhibitsCRAC-mediated Ca²⁺ influx. A series of experiments to test thepossibility that DCPA inhibits STIM 1-Orai I interaction were performed.When ER Ca²⁺ stores are depleted, Ca²⁺ is released from the STIM IEF-hand leading to its aggregation near the plasma membrane instructures often referred to as puncta. Therefore, HEK293 cellstransiently transfected with YFP-STIM I were examined to determine theeffect of DCPA on STIM I puncta formation using fluorescence microscopy.Prior to treatment, all cells displayed a diffuse distribution ofYFP-STIM I. However, depletion of ER Ca²⁺ stores with 2 μM thapsigarginled consistently to extensive puncta formation in control cells within7.5 minutes. In contrast, DCPA treatment dramatically inhibited punctaformation after depleting Ca²⁺ stores over a 16 minute period. Hence, arelatively small number of puncta appeared in specific location, but theoverall distribution of YFP-STIM I in DCPA-pretreated cells wasconsiderably more diffuse than control cells. Considered in combinationwith our CRAC measurements, these findings are consistent with an effectof DCPA on STIM I aggregation and/or interaction with Orai I.

Further, in cells overexpressing both Orai I and the cytosolic fragmentof STIM I (STIM 1 ct), a second CRAC channel modulator,2-aminoethoxydiphenylborate (2-APB), causes STIM 1 ct to translocate tothe plasma membrane and interact with Orai I. To determine if DCPAmodulates interactions between STIM I and Orai I, a similar experimentwas done in the presence or absence of DCPA. YFP-STIM I ct was evenlydistributed throughout the cytoplasm prior to the addition of 2-APB (50μM). As early as 5 seconds after the addition of 2-APB, significanttranslocation of STIM I ct towards the PM could be seen. STIM I ctremains localized to the plasma membrane for at least several minutesafter addition of 2-APB. However, when this experiment was repeated inthe presence of DCPA (100 μM), the ability of 2-APB to induce STIM I cttranslocation to the plasma membrane was dramatically attenuated. Assuch, our data indicate that DCPA inhibits STIM I/Orai I interaction,likely by inhibiting STIM I aggregate formation.

Thus, this invention demonstrates marked changes in Ca²⁺ homeostasisduring RANKL-mediated osteoclast differentiation. Interestingly,coincident with an increase in spontaneous Ca²⁺ oscillations, it wasobserved that a decreased functional activity and expression of the CRACchannel components, Orai I, STIM I, and STIM2. However, this did notreflect a reduced role for Orai I in differentiating osteoclasts; eitherreducing Orai I expression with small interfering RNA or interferingwith its function using DCPA markedly inhibited osteoclastdifferentiation. Indeed, osteoclast differentiation, particularlymultinucleation, occurred at very low rates when Orai expression and/oractivity were suppressed.

The addition of RANKL caused complex changes in Ca²⁺ homeostasis;apparently spontaneous Ca²⁺ oscillations were observed throughout thedifferentiation period, events which coincided with significant changesin the expression and function of the SOCe components STIMI and Orai I(FIG. 1). The role for SOCe in multinucleation is disclosed in thisinvention, however RANKL-induced Ca²⁺ oscillations in differentiatingosteoclasts is known. Ca²⁺ oscillations were attributed to InsP3Ractivity downstream of RANK-dependent ROS production. Ca²⁺ fluxes couldbe observed in cells for as long as 30 minutes after removal ofextracellular Ca²⁺ (data not shown). These observations show thatosteoclast differentiation is highly Ca⁺-dependent, requiring expressionof the InsP3R to mediate the activation of NFATc1, the critical playerin osteoclastogenesis. The present invention discloses that SOCe isspecifically required for terminal differentiation, yet dispensable forearlier events in osteoclast differentiation.

In prior studies where NFATc1 expression was silenced or overexpressed,changes in osteoclast generation correlated directly with similarchanges in the number of TRAP⁺ cells. In the studies reported herein,blockage of Ca²⁺ influx inhibited multinucleation but did not inhibitTRAP activity. A potential explanation for this difference would be thatthe spontaneous and extracellular Ca²⁺-independent Ca²⁺ fluxes that wereobserved throughout the osteoclast differentiation period are sufficientto activate NFATc1 and induce TRAP expression, but insufficient toinduce cell fusion. It is interesting to note that the plasma membraneCa²⁺ channel TRPV4 has also been shown to be important for terminaldifferentiation in osteoclasts. However, trpv4^(−/−) mice differsignificantly in phenotype from Orai I^(−/−) in that trpv4^(−/−) showincreased bone mass attributed to reduced bone resorption, whereas OraiI^(−/−) show poor bone development. Irrespective, consideredcollectively, it seems clear that whereas early events inosteoclastogenesis depend on InsP3R and ROS, osteoclast fusion is highlydependent on extracellular Ca⁺.

Previously, it has been established that DCPA can inhibit store-operatedCa²⁺ entry, although the mechanisms whereby this was achieved wereentirely unclear. In addition to demonstrating its potential as aninhibitor of osteoclastogenesis, it has now been shown that the channelsinhibited by DCPA are specifically CRAC channels. This invention showsthat this inhibition results from interference of STIM-Orai interaction.

FIG. 3 shows the known chemical structure ofN-(3,4-dichlorophenyl)propionamide, also known as “DCPA” or“N-(3,4-dichlorophenyl)propanamide” or “PROPANIL”.

FIG. 4 shows the chemical structures of the more preferred haloanilidecompositions of the present invention, namely,N-(3,4-dibromophenyl)propionamide (also known as “DBPA” or“N-(3,4-dibromophenyl)propanamide”), andN-(3,4-dichlorophenyl)-N-methylpropionamide (also known as“N,N-Me,Propyl-CA” or “N-Methyl-DCPA” or “NMP” orN-(3,4-dichlorophenyl)-N-methylpropanamide”.

FIG. 5 shows the chemical structures of the more preferred haloanilidecompositions of the present invention, namely,N-(3,4-dichlorophenyl)isobutylamide (also known as “N-Isobutyl-CA” or“DNI”), and N-(3,4-dichlorophenyl)-N-methylisobutyramide (also known as“N,N-Me,Isobutyl-CA”).

FIG. 6 shows a graph having the toxicity results of DCPA; DBPA;N,N-Me,Propyl-CA; N-Isobutyl-CA; N,N-Me, Isobutyl-CA; and ethanolcontrol on Jurkat cells exposed 48 hours to each of these compositions,respectively, at the concentrations indicated on the x-axis of thegraph, and analyzed by flow cytometry using the far-red fluorescent DNAbinding probe 7-aminoactinomycin D to indicate dead or late apoptoticcells.

FIG. 7 shows synthesis scheme for the compositions of the presentinvention. In FIG. 7, compositions of the present invention identifiedby numerals 4-7 are prepared by condensation of compounds 1 and 2, neatwith mild heating and methylation (4 and 6, eqn 1). The condensationproducts were isolated by filtration after quenching with water andpurified by recrystallization. Methylation was achieved by forming thesodium salt with NaH, treatment with methyl iodide and recrystallizationof the product.

FIG. 8 shows inhibition of CRAC channels with a composition of thepresent invention, namely, DBPA [ie. N-(3,4-dibromophenyl)propanamide].Jurkat cells, a T cell line that is very sensitive to DCPA, was culturedwith the indicated compounds, DCPA, DBPA, and ethanol (EtOH). CRACchannel activity was monitored using Fura-2 fluorescence using standardknown techniques. Cells were incubated in each composition at 100 uM for10 minutes prior to addition of the SERCA inhibitor thapsigargin (Tg) toinduce SOCe. Decreased CRAC channel activity is clearly evident withDCPA and DBPA. However, DBPA of the present invention does not have theburden of toxicity of DCPA that would accompany further metabolism ofDCPA.

FIG. 9 shows inhibition of CRAC channels with compositions of thepresent invention, namely, DNI, NM Propanil, and DNMNI. Jurkat cells, aT cell line that is very sensitive to DCPA, was cultured with theindicated compositions of the present invention, namely, DNI, NMPropanil, and DNMNI. CRAC channel activity was monitored using Fura-2fluorescence using standard known techniques. Cells were incubated ineach composition at 100 uM for 10 minutes prior to addition of the SERCAinhibitor thapsigargin (Tg) to induce SOCe. Decreased CRAC channelactivity is clearly evident with each of the compositions of the presentinvention. Each of the compositions of the present invention showedsimilar levels of inhibition as the known DCPA. Each of the compositionsof this invention achieve the results achieved with the known DCPA butwithout the burden of toxicity of DCPA that would accompany furthermetabolism of DCPA.

FIG. 10A shows the dose response of a composition of the presentinvention, namely, N-(3,4-dichlorophenyl)-N-methylpropanamide or “NMP”or “N-methyl DCPA” at doses of 3 μM, 10 μM, 15 μM, 60 μM, 100 μM, 150μM, 300 μM, and 1 mM, respectively, which is at least equal to that ofthe known DCPA composition. FIG. 10B shows Store-Operated Calcium Entry(SOCE) inhibition of NMP having a IC₅₀ of 104.2 μM.

It will be appreciated by those persons skilled in the art that thepresent invention shows that the CRAC channel is a Ca²⁺ channel requiredfor normal osteoclast differentiation in vitro. Further, based on thedemonstrated effect of DCPA on SOCe via inhibition of STIM I puncta,siRNA knockdown of Orai I and the lack of an effect of these treatmentson TRAP staining in monocytes after RANKL and m-CSF stimulation, thisinvention discloses that induction of TRAP activity is not dependent onextracellular Ca²⁺. In contrast, multinucleation, another keycharacteristic of osteoclast differentiation, is dependent on functionalCRAC channels. As such, the haloanilide compositions of the presentinvention and methods provided Ca²⁺ channel inhibitors that impact therole of osteoclasts in osteoporosis, osteopetrosis, and bone degradationassociated with arthritis.

These terms and specifications, including examples, serve to describethe invention by example and not to limit the invention. Whereasparticular embodiments of this invention have been described forpurposes of illustration, it will be evident to those persons skilled inthe art that numerous variations of the details of the present inventionmay be made without departing from the invention as defined herein andin the appended claims.

What is claimed is:
 1. A method of inhibiting osteoclast developmentcomprising: administering an effective amount of a haloanilidecomposition or a salt thereof to an osteoclast cell for inhibitingosteoclast development, wherein said haloanilide composition is notN-(3,4-dichlorophenyl)propanamide, and wherein said haloanilide is acomposition of Formula I, or salt thereof, comprising

wherein X is chlorine, wherein R is an ethyl group, and wherein Y is amethyl group.
 2. A method for preventing bone erosion in a patientdiagnosed with arthritis comprising: administering to a patient aneffective amount of a haloanilide composition or salt thereof forpreventing bone erosion in a patient diagnosed with arthritis, whereinsaid haloanilide composition is not N-(3,4-dichlorophenyl)propanamide,and wherein said haloanilide is a composition of Formula I, or saltthereof, comprising

wherein X is chlorine, wherein R is an ethyl group and Y is a methylgroup.